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Significance

Fossils, in addition to documenting the existence of extinct species, can often provide information on the behavior of ancient organisms. The present study describes the fossil of a blood-engorged mosquito in oil shale from northwestern Montana. The existence of this rare specimen extends the existence of blood-feeding behavior in this family of insects 46 million years into the past. Heme, the oxygen-carrying group of hemoglobin in the host’s blood, was identified in the abdomen of the fossil mosquito by nondestructive mass-spectrometry analysis. Although large and fragile molecules such as DNA cannot survive fossilization, other complex organic molecules, in this case iron-stabilized heme, can survive intact and provide information relative to the mechanisms of the fossilization process.

Abstract

Although hematophagy is found in ∼14,000 species of extant insects, the fossil record of blood-feeding insects is extremely poor and largely confined to specimens identified as hematophagic based on their taxonomic affinities with extant hematophagic insects; direct evidence of hematophagy is limited to four insect fossils in which trypanosomes and the malarial protozoan Plasmodium have been found. Here, we describe a blood-engorged mosquito from the Middle Eocene Kishenehn Formation in Montana. This unique specimen provided the opportunity to ask whether or not hemoglobin, or biomolecules derived from hemoglobin, were preserved in the fossilized blood meal. The abdomen of the fossil mosquito was shown to contain very high levels of iron, and mass spectrometry data provided a convincing identification of porphyrin molecules derived from the oxygen-carrying heme moiety of hemoglobin. These data confirm the existence of taphonomic conditions conducive to the preservation of biomolecules through deep time and support previous reports of the existence of heme-derived porphyrins in terrestrial fossils.

Hematophagy is a feeding strategy that has arisen independently numerous times and occurs in five orders of extant insects including fleas (Siphonaptera), lice (Phthiraptera), Lepidoptera, and true bugs [Hemiptera (e.g., Cimicidae or bed bugs)], but is most common in the family Diptera, where it is found in ∼9,000 species in 16 different families (1⇓⇓–4). One extinct family of Cretaceous scorpionflies (Mecoptera), Pseudopolycentropodidae, may have been blood feeding, but this conclusion is controversial (5, 6). The mosquitoes (Culicidae) are by far the most studied hematophagic insects as a consequence of their ability to serve as vectors of widespread diseases such as malaria and yellow fever. As might be expected, the fossil record of hematophagous insects, and mosquitoes in particular, is poor. The majority of all described extinct hematophagous insects are biting midges (Ceratopogonidae) (7). Although there are roughly similar numbers of extant species of Culicidae and Ceratopogonidae, there are ∼200 described extinct species of the latter family, whereas only 25 species of fossil mosquitoes have been described (7⇓–9). One important factor that undoubtedly contributes to this disparity is that, whereas 70% of all fossil mosquito specimens are found in shale, more than 80% of Ceratopogonid fossils are inclusions in amber (10). This difference is the result of a strong taphonomic filter that segregates species and even entire families based on their propensity to populate a specific environmental niche; in this case, a forest of resin-producing trees.

Our ability to identify hematophagous insects is based on the morphology of their mouthparts and their taxonomic affiliations. Fossils that contain direct evidence of hematophagy are extremely rare. Poinar and Poinar have described two species of trypanosome parasites from the gut and proboscis of sandflies (Diptera: Psychodidae) embedded in amber from the Dominican Republic and Myanmar, and Trypanosoma flagellates in a fecal pellet adjacent to an assassin bug (Hemiptera: Reduviidae) in Dominican amber (11⇓⇓–14). Although many trypanosomes are restricted to a single insect host, a few genera are heteroxenous and have a life cycle that requires both blood-sucking insect and vertebrate hosts (15). Given the similarities of the fossilized trypanosomes to known extant heteroxenous species, and the hematophagic lifestyle of the extant relatives of the insect hosts, Poinar has concluded that these fossils represent examples of hematophagy. Even more direct evidence of hematophagy is the observation of nucleated erythrocytes containing putative parasitophorous vacuoles in the gut of an amber-embedded sandfly (16).

Poinar has also reported the presence of Plasmodium sporozoites in the salivary gland and salivary gland ducts of a fossil female mosquito of the genus Culex, some extant species of which are vectors of avian malaria (17, 18). Hematophagy is an obligate lifestyle for female mosquitoes that are blood-feeders (many species are not blood-feeders), as a blood meal is required for the completion of each gonatotrophic cycle and the resultant production of fertile eggs (19). Given the rarity of mosquito fossils, the fossil record’s bias against preservation of mosquitoes in amber, and the fragile nature of blood-engorged mosquitoes, it is not surprising that a fossil of a blood-engorged mosquito has not been described; this despite the popular misconception of dinosaur DNA recovery from blood-engorged mosquitoes in amber popularized by the 1993 film Jurassic Park. Here, we describe a unique fossil of a 46-million-year-old blood-engorged mosquito collected from the Kishenehn Formation in northwestern Montana. The abdomen of the fossil mosquito is shown to contain very high levels of iron, and time-of-flight secondary ion mass spectrometry (ToF-SIMS) data provide unequivocal identification of porphyrin molecules. The combination of these two determinations indicates that the porphyrins are derived from the oxygen-carrying heme moiety of hemoglobin.

Results

To date, a total of 36 specimens of mosquitoes (Diptera: Culicidae) have been collected from the Coal Creek Member of the Kishenehn Formation in northwestern Montana, an emerging Konservat–Lagerstätte of the early Middle Eocene assigned an age of 46 Ma (20). Ten of these specimens have been identified as two new species of the genus Culiseta, Cs. kishenehn and Cs. lemniscata (8). Although the specimens discussed here are not preserved with the detail of the two fossil Culiseta species, the habitus of specimen USNM 559050 is obviously that of a female blood-engorged mosquito with nonplumose antennae and a very dark red/black distended abdomen compared with the nonhematophagous male USNM 559051 (Fig. 1, SI Text, and Fig. S1).

In a study that demonstrated that structures identified as fossilized erythrocytes were in fact diagenetic in origin, it was suggested that heme, localized to specific structures, would constitute incontrovertible evidence for the identification of these cells (21). Because heme consists of both iron and the prosthetic group protoporphyrin, the fossil mosquitoes were analyzed for both. The presence of iron and other elements was determined in situ by use of energy dispersive X-ray spectroscopy (EDS). These analyses found high weight % values of carbon in the abdomen and thorax of the female (66.9% and 51.1%, respectively), whereas the value for the shale matrix was 21.1% (Table 1). Because male mosquitoes do not take blood meals, the abdomen of a fossil male mosquito from the same collection site was analyzed as a control and provided a carbon weight % value of 51.9 Wt %, nearly identical to that of the female thorax. These data suggest that both fossils retain a significant amount of their original compliment of carbon. As might be expected, the abdomen, once laden with protein-rich erythrocytes, is particularly carbon-rich. The weight % value for iron in the abdomen of the female mosquito was 8.97 ± 0.32%, 7.9 and 8.0 times higher than in the thorax of the female and the abdomen of the male mosquito, respectively (Table 1). EDS analysis of purified pig hemoglobin gave a weight % value of iron of 0.33%; the value reported for human hemoglobin is 0.34% (22). The percentage of iron in the abdomen of the female would be expected to increase as other more labile components decayed and were removed especially if the iron were bound in the very stable Fe–porphyrin complex. EDS analysis of permineralized elements suspected to be erythrocytes from the dinosaur Tarbosaurus bataar were reported to have a weight % iron of 8.09% (23). However, iron can be a major diagenetic component of the fossilization process, and high levels of iron, unrelated to a blood meal, have been demonstrated in other fossil insects (24). The iron component of the shales examined in the current study does not appear to be the result of pyrite (FeS2). Although sulfur was present in all samples examined, its concentrations did not correlate stoichiometrically with those of iron. The weight % sulfur varied only slightly, from 1.36% to 1.72% in the thorax and abdomen of the female and the abdomen of the male whereas the respective values for iron were highly disparate (Table 1). ToF-SIMS analyses of the two mosquito specimens detected neither FeS+ nor FeS2+. Neither EDS nor ToF-SIMS analyses detected siderite (FeCO3).

The two fossil mosquitoes were examined for the presence of heme by ToF-SIMS. The ToF-SIMS spectrum obtained from the abdomen of the female was remarkably similar to the spectrum obtained from purified hemoglobin and decidedly different from that obtained from matrix adjacent to the fossil (Fig. 2). Analyses were done at multiple sites on the female abdomen and thorax, the matrix adjacent to the abdomen of the female mosquito, the abdomen and thorax of the male mosquito, and matrix adjacent to the male mosquito (Fig. 3 and Fig. S1). The spectrum characteristic of heme-derived porphyrins was absent at all sites examined except for the female abdomen and analyses at three different sites on the female abdomen produced identical spectra (Fig. 3). Both the female abdomen and hemoglobin control spectra contain a large number of peaks in the mass region between mass/charge ratio (m/z) 350–520, forming Gaussian-like patterns (Fig. 2 and Table S1) separated by mass unit 14 (CH2). This fragmentation pattern, which is the result of Bi+ ion bombardment-mediated fragmentation, is characteristic of porphyrin type structures including chlorophyll and heme and can serve as a fingerprint for identifying these molecules (25⇓⇓⇓⇓⇓–31). The essential identity of the spectra taken from the hemoglobin control and the abdomen of the female mosquito is clearer when the peaks are enlarged (Fig. 2 D and E). Comparison of the exact masses of analyzed peaks with the hemoglobin control and calculated masses of porphyrin derivatives (Table S1), as well as previous ToF-SIMS studies on heme, strongly indicate that the iron is still part of the porphyrin structure (25, 28⇓–30). The most significant difference between the spectra of the female mosquito abdomen and the spectra of the purified pig hemoglobin is the absence of the peak for the intact heme ion [M]+ at m/z 616.18 and the fragment ions centering at m/z 557.14, [M-CH2COOH]+. The lack of these peaks is likely due to partial degradation of the heme molecule in the female mosquito, possibly occurring during diagenesis.

ToF-SIMS (positive detection mode) spectra of heme-derived porphyrins in the abdomen of the female blood-engorged mosquito (USNM 559050) and controls. (A) A spectrum of the abdomen of the blood-engorged mosquito. (B) A spectrum of the heme and heme-derived porphyrins in purified pig hemoglobin. Peaks m/z 455.04, m/z 469.05 and m/z 483.08 are three of numerous peaks present in both the abdomen of the female mosquito and pure hemoglobin (see list of heme-derived phorphyrins and their possible structures in Table S1). Intact heme has a mass/charge ratio (m/z) of 616.18. (C) A spectrum obtained from the matrix adjacent to the abdomen of the female mosquito. Peaks that denote specific porphyrins as well as the distinctive porphyrin spectral pattern are absent. (D–F) Enlarged insets of mass region 460.4–474.4 of the spectra on the left (A–C) further illustrate the Gaussian distribution of peaks and near identity between the abdominal and hemoglobin control spectra. See Table S1 for the proposed origins of peaks 467.03 and 469.05.

ToF-SIMS spectra from different areas of the female (USNM 559050) and male (USNM 559051) mosquitoes. (A) Spectra taken from three different areas of the abdomen of the female mosquito. (B) Control spectra obtained from two different areas of matrix adjacent to the female mosquito, two different areas of the thorax of the female mosquito, two different areas of the abdomen of the male mosquito and a single spectrum of the matrix adjacent to the male mosquito.

A thin layer of potassium aluminum silicate overlaying the fossils was removed before analysis. Scanning electron microscopy of the female abdomen clearly distinguishes between the opaque silicate and the underlying smooth carbonaceous fossil (Fig. 4A). ToF-SIMS ion images show colocalization of iron, several different porphyrins, and CN− derived from the heterocyclic pyrrole groups of the porphyrin within exposed areas of the abdomen of the female mosquito (Fig. 4 D–F). These areas are easily distinguishable from the surrounding silicate matrix by the high silicon and potassium content of the matrix (Fig. 4 B and C). Apparent colocalization of Fe and porphyrins in ToF-SIMS ion images supports spectral evidence of the Fe being bound to the porphyrin structures. Variability in the area exposed for analysis and the inherent differences in heme concentration between the standard and the fossil account for the peak intensity differences in Figs. 2 and 3.

Colocalization of heme-derived porphyrins and iron. (A) A scanning electron micrograph (backscattered electron mode) of a portion of the abdomen of the blood-engorged female mosquito (USNM 559050). The actual fossil surface is black with multiple minute cracks due to dehydration. Large portions of the fossil surface are covered by an opaque layer of potassium magnesium aluminum silicate as indicated by ToF-SIMS ion image localization of Si+ (m/z 27.98; B) and K+ (m/z 38.97; C). (D–F) Colocalization of heme-derived porphyrins (m/z 460.40–474.40), CN− (m/z 26.00) characteristic of the heterocyclic pyrrole groups of porphyrin, and Fe+ (m/z 55.93), respectively, in the area depicted in A. (Scale bar, 50 μm.)

Discussion

The preservation of fossil female mosquito USNM 559050 was an extremely improbable event. The insect had to take a blood meal, be blown to the water’s surface, and sink to the bottom of a pond or similar lacustrine structure to be quickly embedded in fine anaerobic sediment, all without disruption of its fragile distended blood-filled abdomen. This fossil has provided a unique opportunity to ask whether or not a portion of the hemoglobin molecule could be preserved after tens of millions of years; heme was the most obvious target for our analysis. Detection of heme-derived porphyrins in the female specimen confirms that it is indeed a blood-engorged mosquito and provides direct evidence of hematophagy in the fossil record. The two species of mosquitoes known from the Kishenehn basin, Culiseta kishenehn and Cs. lemniscata, most closely resemble extant species of the subgenera Climacura and Culicella, respectively (8). Both subgenera are small, with 5 and 14 species, respectively, and both contain species that feed almost exclusively on birds [e.g., Culiseta (Culicella) morsitans and Culiseta (Climacura) melanura] (32). The host of the blood-engorged mosquito described herein, which is similar to Cs. lemniscata (SI Text), is unknown.

Porphyrins are energetically very stable molecules and have been shown to be preserved through geological time as common components of many oil shales (28, 29). Although the majority of such geoporphyrins are derived from chlorophyll and bacteriochlorophyll, some are thought to be derived from either heme or its precursors found in microbial cytochromes and related proteins. A microbial origin of the porphyrins found in the blood-engorged mosquito is unlikely based on the following observations: (i) Scanning electron microscopy did not reveal any microbial structures, either intact or fragmentary, on the surface of the fossil. (ii) If microbes were the source of the porphyrin, their growth would not be expected to be completely limited to a portion (i.e., the abdomen) of the body of the fossil mosquito. (iii) The anaerobic environment required for the preservation of the Kishenehn fossil insects would have supported only anaerobic microbes. Because such anaerobic organisms have lost their ability to use molecular oxygen as a terminal electron transporter in anaerobic environments, they express very low levels of porphyrin-containing cytochromes (33).

Reports of the identification of hemoglobin and/or heme in fossils are few in number, equivocal and do not localize these molecules in situ. Blood vessels isolated from trabecular bone of the dinosaur Brachylophosaurus canadensis bound hemoglobin-specific antiserum in an immunosorbant assay, albeit with a signal only twice that of background levels (34). In immunoblot assays, antiserum raised against extracts of Tyrannosaurus rex trabecular bone reacted with purified hemoglobin but antiserum against hemoglobin did not react with extracts of B. canadensis bone (34, 35). Both NMR and Raman spectroscopy have been applied to extracts of T. rex trabecular bone tissue, and data from both techniques were suggestive of the presence of heme and/or porphyrin (35). The data reported herein provide incontrovertible documentation of the presence of heme- and arguably hemoglobin-derived porphyrins in a 46-million-year-old compression fossil and localize the porphyrins to a specific anatomical structure within that fossil.

As previously suggested, ToF-SIMS has significant potential for the unequivocal detection and nondestructive in situ localization of heme and related porphyrins in fossilized life forms (29). Although not readily applicable to amber inclusions, wider application of this technology to compression fossils may lead to the identification of other biomolecules and provide insight as to their heretofore unsuspected preservation.

Materials and Methods

Specimens are housed at the Department of Paleobiology, National Museum of Natural History (NMNH), Smithsonian Institution (Washington, DC). Fossils were immersed in 95% ethanol for examination and photographed with an Olympus SZX12 microscope equipped with a Q-Color5 Olympus camera. Image-Pro Plus 7.0 software was used to capture and record the images and measurements. A thin layer of silicate covering the fossil mosquito specimens was removed through use of a Faber–Castell eraser pencil (no. 185698) and/or a Becton Dickinson 1 cc insulin syringe with attached 29-gauge needle (no. 309311). Samples were rinsed with ethanol and mounted for in situ ToF-SIMS analysis in a laminar flow hood. Microanalysis and imaging were performed at the NMNH Department of Mineral Sciences on uncoated samples using an FEI NOVA NanoSEM 600 FEG Variable Pressure Analytical Scanning Electron Microscope (SEM) outfitted with a ThermoNoran energy dispersive spectrometer (EDS) and a ToF-SIMS IV (ION-TOF) spectrometer. Standardless EDS analyses were performed at 15 kV and 1–2 nA and have an analytical uncertainty of less than 5%. ToF-SIMS ion imaging was done using a 25 keV Bi3+ beam at a pulsed current of 0.3 pA, rastered over an area of ∼300 × 300 µm for 180–600 s. The accumulated primary ion dose never exceeded 1.25 × 1010 ions per cm2, which is below the static limit (the point where the same location is statistically sampled more than once) of 1012 ions per cm2 for organic molecules (36). Analyses were performed with the instrument optimized for high mass resolution (bunched mode: m/∆m of at least ∼5,000 at m/z 30). Pig hemoglobin (SigmaAldrich H4131-1G), dissolved in water at 5 mg/mL and evaporated onto the surface of an aluminum stub and a glass slide at 55 °C, was used as a positive control in EDS and ToF-SIMS experiments, respectively.

Acknowledgments

We thank Kurt, Leona, and Norm Constenius for the gift of their collection of fossil insects from the Kishenehn Formation to the NMNH. We also thank Conrad Labandeira for reviewing the manuscript, Finnegan Marsh for administrative support, Keana Scott for her input with EDS, and three anonymous reviewers for constructive input. This work was funded in part by the Deep Carbon Observatory (A. Steele, Principle Investigator), the Postdoctoral fellowship program of Geophysical Laboratory, Carnegie Institution of Washington, and the Swedish National Space Board (Contract 121/11). This is Contribution 283 of the Evolution of Terrestrial Ecosystems Consortium of the US National Museum.